Expression of gene products from genetically manipulated strains of bordetella

Abstract

An expression system for expressing gene products from recombinant Bordetella strains and specific nucleic acid molecules useful in transforming Bordetella strains for such expression are described. A nucleic acid molecule may comprise a Bordetella promoter operatively coupled to a heterologous gene encoding a non-Bordetella gene product with the heterologous gene transcriptionally regulated by the Bordetella promoter. The nucleic acid molecule may further comprise a further nucleic acid molecule encoding a leader sequence for secretion of the non-Bordetella gene product. Another nucleic acid molecule may comprise a Bordetella promoter coupled to a nucleic acid sequence encoding a non-Bordetella leader sequence for secretion of a gene product, which may be a Bordetella gene product or a non-Bordetella gene product.

What we claim is: 
     
       1. A nucleic acid molecule, comprising a Bordetella promoter selected from the group consisting of the tox, prn and fha promoter operatively coupled to a heterologous gene encoding a non-Bordetella gene product, wherein the heterologous gene is transcriptionally regulated by the Bordetella promoter. 
     
     
       2. The nucleic acid molecule of claim 1 wherein the non-Bordetella gene product is selected from the group consisting of an enzyme, an antigen, an immunogen, an allergen, an enzyme inhibitor, a hormone, a lymphokine, an immunoglobulin or fragment thereof, a toxin, a toxin subunit, a mammalian protein, a structural protein and a receptor. 
     
     
       3. The nucleic acid molecule of claim 1 wherein the non-Bordetella gene product is a cholera toxin molecule. 
     
     
       4. The nucleic acid molecule of claim 3 wherein the cholera toxin molecule is the B subunit thereof. 
     
     
       5. A recombinant strain of Bordetella containing the nucleic acid molecule of claim 1 and expressing said non-Bordetella gene product. 
     
     
       6. A nucleic acid molecule, comprising a Bordetella promoter selected from the group consisting of the tox, prn and fha promoter operatively coupled to a heterologous gene encoding a non-Bordetella gene product and a nucleic acid sequence encoding a leader sequence for secretion of the non-Bordetella gene product, wherein the heterologous gene is transcriptionally regulated by the Bordetella promoter. 
     
     
       7. The nucleic acid molecule of claim 6 wherein the leader sequence is a leader sequence of a Bordetella protein or subunit thereof or a fragment or analog of the Bordetella protein leader sequence retaining secretion-mediating properties. 
     
     
       8. The nucleic acid molecule of claim 7 wherein the leader sequence is selected from the group consisting of the Bordetella pertactin leader sequence and a pertussis toxin subunit leader sequence. 
     
     
       9. The nucleic acid molecule of claim 7 wherein the heterologous gene encodes a cholera toxin molecule. 
     
     
       10. The nucleic acid molecule of claim 9 wherein the cholera toxin molecule is the B subunit thereof. 
     
     
       11. The nucleic acid molecule of claim 6 wherein the leader sequence is a leader sequence of a non-Bordetella protein or subunit thereof or a fragment or analog of the non-Bordetella protein leader sequence retaining secretion-mediating properties. 
     
     
       12. The nucleic acid molecule of claim 11 wherein the non-Bordetella gene product is a secreted gene product and the non-Bordetella leader sequence is the native leader sequence of said secreted gene product. 
     
     
       13. The nucleic acid molecule of claim 12 wherein the secreted gene product is a cholera toxin molecule. 
     
     
       14. The nucleic acid molecule of claim 13 wherein the cholera toxin molecule is the B subunit thereof. 
     
     
       15. The nucleic acid molecule of claim 6 wherein said Bordetella promoter is the fha promoter of Bordetella pertussis and the leader sequence for secretion of the non-Bordetella gene product is the leader sequence of the PRN protein. 
     
     
       16. A nucleic acid molecule selected from the group consisting of toxp/CTB-L/ctb, fhap/CTB-L/ctb, toxp/S1-L/ctb, toxp/PRN-L/ctb, fhap/S1-L/ctb and fhap/PRN-L/ctb wherein: toxp is the tox promoter of Bordetella pertussis,   fhap is the fha promoter of Bordetella pertussis,   CTB-L is a nucleic acid sequence encoding a leader sequence for cholera toxin subunit,   S1-L is a nucleic acid sequence encoding a leader sequence for Bordetella pertussis toxin S1 subunit,   PRN-L is a nucleic acid sequence encoding a leader sequence for pertactin, and   ctb is a heterologous gene encoding a cholera toxin B-subunit.   
     
     
       17. The nucleic acid molecule of claim 16 which is fhap/PRN-L/ctb. 
     
     
       18. A nucleic acid molecule comprising a Bordetella promoter coupled to a nucleic acid sequence encoding a non-Bordetella leader sequence for secretion of a gene product, operatively coupled to acid leader sequence. 
     
     
       19. The nucleic acid molecule of claim 18 wherein the non-Bordetella leader sequence is selected from bacterial leader sequences, eukaryotic leader sequences and viral leader sequences. 
     
     
       20. The nucleic acid molecule of claim 19 wherein the non-Bordetella leader sequence is the leader sequence for the cholera toxin B subunit. 
     
     
       21. A recombinant strain of Bordetella containing the nucleic acid molecule of claim 18 and secreting said gene product. 
     
     
       22. A method of expression of a gene product, which comprises culturing a recombinant strain of claim 21, and isolating the expressed gene product. 
     
     
       23. A nucleic acid molecule comprising: a first DNA sequence corresponding to a 5' flanking sequence of a selected Bordetella gene and disposed at the 5' end of the nucleic acid molecule,   a Bordetella promoter selected from the group consisting of the tox, prn and fha promoter operatively coupled to a heterologous gene encoding a non-Bordetella gene product,   a nucleic acid sequence encoding a leader sequence for secretion of the non-Bordetella gene product, the heterologous gene being transcriptionally regulated by the Bordetella promoter, and   a second DNA sequence corresponding to a 3' flanking sequence of the selected Bordetella gene and disposed at the 3' end of the nucleic acid molecule,   said first and second DNA sequences permitting specific integration of the nucleic acid molecule into the Bordetella genome at a locus corresponding to the selected Bordetella gene.   
     
     
       24. The nucleic acid molecule of claim 23 wherein said Bordetella promoter is the fha promoter of Bordetella pertussis and the leader sequence for secretion of the non-Bordetella gene product is the leader sequence of the PRN protein. 
     
     
       25. The nucleic acid molecule of claim 24 wherein said first and second DNA sequences correspond respectively to 5' and 3' flanking sequences of the fha gene of Bordetella pertussis to permit specific utilization of the nucleic acid molecule into the presence of Bordetella pertussis at the locus corresponding to the fha gene. 
     
     
       26. A plasmid adapted for transformation of a Bordetella strain comprising the nucleic acid molecule of claim 23. 
     
     
       27. A plasmid adapted for transformation of a Bordetella strain comprising the nucleic acid of claim 25. 
     
     
       28. A recombinant strain of Bordetella having a nucleic acid molecule integrated into the genome thereof at the locus of a selected Bordetella gene selected from the group consisting of the tox operon, prn gene and fha gene, said nucleic acid molecule comprising a 5' flanking sequence of said selected Bordetella gene, a Bordetella promoter selected from the group consisting of the tox, prm and fha promoters operatively coupled to a nucleic acid sequence encoding a leader sequence for secretion of a non-Bordetella gene product; a heterologous gene encoding a non-Bordetella gene product, wherein a heterologous gene is transcriptionally regulated by the Bordetella promoter, and a 3' flanking sequence of said selected Bordetella gene, said recombinant strain secreting said non-Bordetella gene product. 
     
     
       29. The strain of claim 28 wherein the Bordetella strain is a selected from the group consisting of a B. pertussis strain, a B. parapertussis strain, a B. bronchiseptica strain and a B. avium strain. 
     
     
       30. The strain of claim 29 wherein the Bordetella strain is a strain of B. pertussis. 
     
     
       31. A method of expression of a non-Bordetella gene product, which comprises culturing a recombinant strain of claim 28, and isolating the expressed non-Bordetella gene product.